Process for isomerizing glucose to fructose

ABSTRACT

An enzymatically active product for use in isomerization of glucose into fructose is provided. The active product comprises an organic polymeric material predominantly comprised of a monovinyl aromatic compound polymer having β-aminopropionamidomethyl group as side chains represented by the formula I: ##STR1## wherein R 1  is selected from H, C(1-6) alkyl and C(2-6) hydroxyalkyl, R 2  is selected from C(1-6) alkyl, C(2-6) hydroxyalkyl, ##STR2## (where X, Z 1  and Z 2  are selected from H and C(1-6) alkyl, and n is from 2 to 6), or R 1  and R 2  form together with the N atom, to which R 1  and R 2  are bonded, heterocyclic structure of the formula: ##STR3## where A is --CH 2  --, --O-- or --NR 6  -- (wherein R 6  is H or C(1-6) alkyl), and R 3 , R 4  and R 5  are selected from H and methyl. The organic polymeric material has glucose isomerase immobilized with the β-aminopropionamidomethyl group side chain of the organic polymeric material.

This is a continuation of application Ser. No. 114,270 filed Jan. 22,1980, now U.S. Pat. No. 4,275,156, issued June 23, 1981.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

This invention relates to a process for isomerizing a glucose-containingsolution to convert at least a part of the glucose to fructose.

(2) Description of the Prior Art

Glucose isomerase is an enzyme capable of converting glucose intofructose and vice versa and is used for producing fructose from glucose.

Many proposals have been made to stabilize and/or to immobilize glucoseisomerase for the multiple reuse thereof. For example, U.S. Pat. Nos.3,708,397 and 3,788,945 teach the immobilization of glucose isomerasewith DEAE, TEAE or ECTEOLAcellulose. These celluloses are used in theform of a microgranule, fine fiber or a cake. These forms are notconvenient to handle. Furthermore, when these celluloses are packed in acolumn, they tend to be densified during the use, and hence, cause alarge pressure loss and prevent the passage of a glucose substratesolution. Japanese Laid-open Patent Application 53,582/1975 disclosesthe immobilization of glucose isomerase with a macroreticulated orporous anion exchange resin. However, the isomerase immobilized anionexchange resin exhibits an undesirably low enzymatic activity and isalso poor in the retention of activity. Japanese Laid-open PatentApplications 1181/1973 and 92,277/1974 teach that a homogenizedmicroorganism cell concentrate containing ruptured cells is treated withglutaraldehyde to form a crosslinked coherent solid product. Thiscrosslinked product is also not satisfactory in its enzymatic activity.Furthermore, once the enzymatic activity of the crosslinked productdecreases due to repeated use, it is difficult or even impossible toregenerate its enzymatic activity.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a process forisomerizing a glucose-containing solution to convert at least a part ofthe glucose to fructose by a process of isomerization.

Another object of the present invention is to provide a process forproducing an isomerized glucose-containing solution, containingfructose, which is suitable for subsequent separation.

Still another object of the present invention is to provide such aprocess which enhances fructose productivity.

Other objects and advantages of the present invention will be apparentfrom the following description.

The enzymatically active product which is useful in the process of theinvention for use in isomerization of glucose into fructose comprises anorganic polymeric material comprised of at least 50% by weight, based onthe weight of the organic polymeric material, of a monovinyl aromaticcompound in polymerized form. The polymerized monovinyl aromatic compundhas a β-aminopropionamidomethyl group as a side chain represented by theformula I: ##STR4## wherein R₁ is selected from hydrogen, an alkyl grouphaving 1 to 6 carbon atoms and a hydroxyalkyl group having 2 to 6 carbonatoms; R₂ is selected from an alkyl group having 1 to 6 carbon atoms, ahydroxyalkyl group having 2 to 6 carbon atoms; ##STR5## (where X, Z₁ andZ₂ are selected from hydrogen and an alkyl group having 1 to 6 carbonatoms, and n is an integer of from 2 to 6); or R₁ and R₂ form togetherwith the nitrogen atom, to which R₁ and R₂ are bonded, a heterocyclicstructure represented by the formula ##STR6## where A is --CH₂ --, --O--or --NR₆ -- (R₆ is H or an alkyl group having 1 to 6 carbon atoms); andR₃, R₄ and R₅ may be the same as or different from each other and areselected from hydrogen and a methyl group. The organic polymericmaterial has glucose isomerase immobilized with theβ-aminopropionamidomethyl group side chain of the organic polymericmaterial.

The enzymatically active product of the invention is prepared by aprocess which comprises the steps of (a) treating an article of anorganic polymeric material comprised of at least 50% by weight, based onthe weight of the organic polymeric material, of a monovinyl aromaticcompound in polymerized form, with an acrylamidomethylating agentrepresented by the formula II: ##STR7## wherein R₃, R₄ and R₅ are asdefined above with respect to the formula I, and Z is

    --OR.sub.6

where R₆ is selected from hydrogen, an alkyl group having 1 to 6 carbonatoms and ##STR8## (R₇ is an alkyl group having 1 to 6 carbon atoms), or##STR9## where R₃, R₄ and R₅ are as defined above; (b) treating thearticle with an amino compound represented by the formula III: ##STR10##wherein R₁ and R₂ are as defined above with respect to the formula I,thereby introducing to the polymerized monovinyl aromatic compound aβ-aminopropionamidomethyl group as a side chain represented by theabove-mentioned formula I, and; then, (c) bringing the article intocontact with a glucose isomerase-containing solution or dispersion toimmobilize glucose isomerase with said side chain of the polymerizedmonovinyl aromatic compound.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The organic polymeric material, with which glucose isomerase is to beimmobilized, in the practice of the process of this invention, may be ahomopolymer of a monovinyl aromatic compound or a copolymer (including agraft copolymer) comprised of at least 50% by weight of units derivedfrom a monovinyl aromatic compound and not more than 50% by weight of atleast one other copolymerizable monomers. The monovinyl aromaticcompound used includes, for example, styrene, α-methystyrene,vinyltoluene, vinylxylene and chlorostylene. The copolymerizablemonomers used include, for example, methyl acrylate, methylmethacrylate, acrylonitrile, acrylamide, vinyl chloride and vinylidenechloride.

The above-mentioned organic polymeric material predominantly comprisedof the monovinyl aromatic compound in polymerized form (which materialis hereinafter referred to as "monovinyl aromatic compound polymer" forbrevity) may be employed in combination with other polymeric materials.The polymeric materials used are those which are miscible butsubstantially incompatible with the monovinyl aromatic compound polymer.Such polymeric materials include, for example, polyolefins, polyamides,polyesters and their copolymers. The organic polymeric materialpredominantly comprised of the monovinyl aromatic compound and the otherpolymeric material may be used either as a blend or so that thesepolymeric materials form discrete portions of the article with whichglucose isommerase is immobilized. The proportion of the monovinylaromatic compound polymer to the other polymeric material is notparticularly limited, provided that the monovinyl aromatic compoundpolymer is exposed to the surface of the article, with which theisomerase is immobilized, to an extent such that the monovinyl aromaticcompound polymer occupies at least about one half of the entire surfacearea of the article.

The shape of the article, with which glucose isomerase is immobilized,is not particularly limited, but the article may usually be in the formof a fiber, a particulate or a film. One preferable form of the articleis a fiber comprised of the monovinyl aromatic compound polymer andanother fiber-forming organic polymeric material. The fiber may beeither a blend fiber made from a uniform blend of the two polymericmaterials, or a core-sheath type or islands-in-a-sea type compositefiber, the sheath or sea ingredient being predominantly comprised of themonovinyl aromatic compound polymer and the core or island ingredientbeing comprised of the fiber-forming organic polymeric material. Ofthese fibers, the blend fiber and the islands-in-a-sea type compositefiber are most preferable because of their good resistance toseparation. It is particularly preferable that the number of islands ina sea in the cross-section of the islands-in-a-sea type composite fiberbe at least 5.

In the blend fiber, the amount of the fiber-forming organic polymericmaterial should preferably be less than 50% by weight, based on thetotal weight of the fiber-forming organic polymeric material and themonovinyl aromatic compound polymer. When the amount of thefiber-forming organic polymeric material exceeds 50% by weight, theenzymatically active product resulting from the blend fiber cannotimmobilize the desired amount of glucose isomerase. There is noparticularly critical lower limit for the amount of the fiber-formingorganic polymeric material, but it is preferable to use from 20% to 40%by weight of the fiber-forming organic polymeric material for improvingthe mechanical strength of the fiber and the durability thereof.

In the core-sheath type or islands-in-a-sea type composite type, theamount of the sheath or sea ingredient predominantly comprised of themonovinyl aromatic compound polymer should preferably be in the range offrom about 10 to 90% by weight, more preferably from about 20 to 80%,based on the weight of the composite fiber. When the amount of the coreor island ingredient is less than about 10% by weight, the compositefiber becomes poor in mechanical strengths. In contrast, when the amountof the core or island ingredient exceeds about 90% by weight, theenzymatically active product resulting from the composite fiber becomespoor in the capacity of immobilizing glucose isomerase. The sheath orsea ingredient of the composite fiber may be a mixture of the monovinylaromatic compound polymer and the fiber-forming organic polymericmaterial. In general, when the relative proportion of the fiber-formingorganic polymeric material is large, the composite fiber becomesdensified and, therefore, exhibits enhanced durability, mechanicalstrengths and resistance to separation, but the enzymatically activeproduct resulting therefrom is poor in the capacity of immobilizingglucose isomerase. Thus, the amount of the fiber-forming organicpolymeric material should preferably be less than about 50% by weight,more preferably in the range of from 5 to 40% by weight, based on theweight of the sheath or sea ingredient.

Both the blend fiber and the composite fiber may have either a circularcross-section or a non-circular cross-section. The fiber having anon-circular cross-section advantageously has a larger surface area thanthat of the circular sectional fiber. The blend fiber and the compositefiber may be of a porous structure, which has a far larger surface areathan that of a non-porous structure.

The fineness of the blend fiber and the composite fiber is usually inthe range of from about 0.01 to 500 deniers, preferably from about 0.1to 50 deniers. When the fineness of the fiber is too small, the fiberpossesses poor mechanical strengths and is liable to be broken into fineparticles, and thus, is difficult to handle. In contrast, when thefineness of the fiber is too large, the enzymatically active productresulting from the fiber becomes poor in the capacity of immobilizingglucose isomerase.

The blend fiber and the composite fiber may be used in various forms,such as, for example, filament yarns, staple fibers, a needle-punchedfelt and other non-woven fabrics, woven fabrics, knitted fabrics andbattings.

The monovinyl aromatic compound polymer has as a side chain theβ-aminopropionamidomethyl group of the formula I, hereinbeforementioned. The amount of the β-aminopropionamidomethyl group ispreferably at least about 0.5 meq. more preferably in the range of fromabout 2.0 to 5.0 meq, per g of the monovinyl aromatic compound polymer.When the amount of the β-aminopropionamidomethyl group is less thanabout 0.5 meq/g, the monovinyl aromatic compound polymer is incapable ofimmobilizing the desired amount of glucose isomerase. Although there isno particularly critical upper limit for the amount of theβ-aminopropionamidomethyl group, it is generally difficult to introducemore than about 5.0 meq/g, of the β-aminopropionamidomethyl group intothe monovinyl aromatic compound polymer.

The enzymatically active product of the invention is prepared by thefollowing process. In the first step, an article comprised of either themonovinyl aromatic compound polymer or a combination of the monovinylaromatic compound polymer and the other polymeric material is treatedwith an acrylamidomethylating agent of the formula II, hereinbeforementioned, whereby acrylamidomethyl groups are introduced as side chainsto the monovinyl aromatic compound polymer.

As hereinbefore mentioned, the shape of the article to be treated withthe acrylamidomethylating agent is not particularly limited, but thearticle is preferably in the form of a fiber comprised of the monovinylaromatic compound polymer and another fiber-forming organic polymericmaterial. The fiber may be either a blend fiber, or a core-sheath typeor islands-in-a-sea type composite fiber, the sheath or sea ingredientbeing predominantly comprised of the monovinyl aromatic compound polymerand the core or island ingredient being comprised of the fiber-formingorganic polymeric material. The blend fiber and the composite fiber maybe prepared by a conventional procedure. The monovinyl aromatic compoundpolymer used for the preparation of the blend or composite fiberpreferably possess an intrinsic viscosity of from 0.5 to 5, as measuredin toluene, at 25° C. Both the blend fiber and the composite fiber maybe used in various forms as hereinbefore mentioned.

The treatment of the above-mentioned article with theacrylamidomethylating agent is usually effected in the presence of aswelling agent capable of swelling the monovinyl aromatic compoundpolymer and in the presence of an acid catalyst. The swelling agent usedincludes, for example, halogenated hydrocarbons, such asdichloromethane, carbon tetrachloride, dichloroethane,sym-tetrachloroethane and tetrachloroethylene, and; nitratedhydrocarbons, such as 1- or 2-nitropropane, nitroethane andnitrobenzene. These swelling agents may be used either alone or incombination. Of these swelling agents nitrated hydrocarbons arepreferable.

The acid catalyst used includes, Friedel-Crafts catalysts such as,aluminum chloride, tin tetrachloride, ferric chloride and zinc chloride;strong organic acids, such as aliphatic sulfonic acids (e.g.methanesulfonic acid) and aromatic sulfonic acids (e.g. benzenesulfonicacid and toluenesulfonic acid), and; strong inorganic acids, such assulfuric acid (particularly concentrated and fuming sulfuric acids). Ofthese acid catalysts, sulfuric acid is preferable.

The acrylamidomethylating agent represented by the formula II,hereinbefore mentioned, includes, for example, N-methylolacrylamide andits carboxylic acid esters and alkyl ether derivatives;N-methylolmethacrylamide and its carboxylic acid esters, and alkyl etherderivatives, and; N,N'-(oxydimethylen)bisacrylamide. Theseacrylamidomethylating agents may be used either alone or in combination.The amount of the acrylamidomethylating agent used should preferably besuch that the resulting article contains from about 0.5 to 5.0 meq.,more preferably from about 2.0 to 5.0 meq., per gram of the monovinylaromatic compound polymer.

The acrylamidomethylating reaction may be preferably carried out at atemperature of from about 0° C. to about 60° C., more preferably fromabout 15° C. to about 30° C.

Prior to or during the acrylamidomethylating treatment, the article maybe subjected to a crosslinking treatment wherein a crosslinking agent,such as formaldehyde, is used. The crosslinking treatment enhances themechanical strengths of the article. It should be noted, however, thatthe crosslinking treatment reduces the capacity of the resulting productto immobilize glucose isomerase, and therefore, the degree ofcrosslinking should be to a minor extent.

The acrylamidomethylated product is then treated with an amino compoundof the formula III, hereinbefore mentioned, whereby the acrylamidomethylgroups are converted to β-aminopropionamidomethyl groups. The aminocompound used is not particularly limited, provided that the aminocompound is capable of reacting with the acrylamidomethyl group, therebyconverting the group to a β-aminopropionamidomethyl group. Preferableamino compounds are organoamino compounds (including multiaminocompounds) having at least one primary or secondary amino group andforming little or no crosslinking. Such amino compounds include, forexample, dimethylamine, diethylamine, dipropylamine, dibutylamine,dihexylamine, dipropanolamine, N-methylaminoethanol,N-methylbenzylamine, N,N-diethyl-N'-methylethylenediamine, methylamine,ethylamine, propylamine, butylamine, hexylamine, propanolamine,N,N-dimethylaminopropylamine, ethylenediamine, hexamethyldiamine,N,N-diethylethylenediamine, morpholine, piperidine and piperazine. Theseamino compounds may be used either alone or in combination.

The aminating treatment is usually effected in the presence of a solventcapable of dissolving the amino compound. The solvent used includes, forexample, water; lower alcohols, such as methanol, ethanol and n-butanol,and; ethers, such as dioxane and tetrahydrofuran. The reactiontemperature may be in the range of from room temperature to the refluxtemperature. The reaction pressure may be normal. However, in the casewhere the amino compounds used has a low boiling point, the reactionpressure may be superatmospheric in order to shorten the reaction time.

The aminated product is then brought into contact with a glucoseisomerase-containing solution or dispersion, whereby glucose isomeraseis immobilized with the β-aminopropionamidomethyl group side chains ofthe aminated product.

Glucose isomerase can be obtained from various microorganisms whichinclude, for example, actinomycetes, such as Streptomycesphaseochromogenus and Streptomyces albus, and; bacteria, such asBacillus coagulans, Bacillus megatherium, Lactobacillus brevis, thePseudomonas genus and the Aerobacter genus. Glucose isomerase may beused either in the form of a suspension containing ruptured cell pieces,extracted from microorganism cells, or in the form of a solution whichis obtained by removing the ruptured cell pieces from theabove-mentioned suspension by, for example, centrifuging or filtration,or which is obtained by refining the ruptured cell piece-removedsolution.

The pH value of the glucose isomerase-containing solution or suspensionmay usually be in the range of from about 4 to 12. It is preferable,however, that the pH value be adjusted to the range of from about 5 to 9in order to immobilize the desired large amount of glucose isomerasewith the aminated product having β-aminopropionamidomethyl groups.

The procedure, by which the aminated product is brought into contactwith the glucose isomerase-containing suspension or solution, is notparticularly critical, but may be similar to that popularly employed inconventional ion exchange treatments. For example, the aminated productis immersed in the glucose isomerase-containing solution or dispersion,if desired while being stirred, and then, the product is washed withwater. Alternatively, the glucose isomerase-containing solution orsuspension is passed through a column packed with the aminated productin a fixed bed system, and then, the product is washed with water.

The β-aminopropionamidomethyl groups present in the aminated product maybe either in the form of a salt, or a free form or a form bufferizedwith a suitable buffer. The aminated product having the freeβ-aminopropionamidomethyl groups preferably possesses a water content offrom about 0.5 to 3. The water content usually varies depending upon thestructure of the aminated product, the amount of theaminopropionamidomethyl groups present in the aminated product and thedegree of crosslinking. When the water content is too small, it isdifficult to immobilize the desired amount of the isomerase. Incontrast, when the water content is too large, it becomes difficult tohandle the aminated product.

The period of time and the temperature for the immobilizing treatmentmay suitably be determined so that the amount of glucose isomeraseimmobilized is as large as possible. In general, the amount of glucoseisomerase immobilized may be varied in the range of from 2,000 to 50,000U, particularly from 5,000 to 30,000 U, expressed in terms of theactivity, per gram of the enzymatically active product.

In order to enhance the degree of activity retention of the resultingenzymatically active product, the isomerase-immobilized product may betreated with a solution containing a crosslinking agent, whereby theimmobilized glucose isomerase is crosslinked with the crosslinkingagent. Instead of treating the isomerase-immobilized product, thecrosslinking agent may be incorporated in the glucoseisomerase-containing solution used for immobilization. The crosslinkingagent used is one which is capable of crosslinking a protein and ispopularly called a multi-functional protein modifier. Such acrosslinking agent includes, for example, polyglutaraldehydes, such asglutaraldehyde dialdehyde starch, and; polyisocyanates, such as tolylenediisocyanate and hexamehtylene diisocyanate. When the degree ofcrosslinking is too large, the enzymatic activity of the product isliable to be low, although the degree of activity retention is high.Therefore, consideration should be given to the concentration of thecrosslinking agent in the treating solution, the pH of the treatingsolution, and the treating temperature and time.

The enzymatically active product, particularly in the form of the blendfiber or the composite fiber, has the following advantages.

(i) The fibrous product has an enhanced activity per unit weight of theproduct.

(ii) The fibrous product exhibits a good retention of activity.

(iii) The fibrous product exhibits high mechanical strengths, gooddurability and good separation resistance.

(iv) The fibrous product can be used in an arbitrary form.

(v) The fibrous product has a low water content and is easy to handle.

(vi) The fibrous product can be produced by a simple procedure at a lowproduction cost.

(vii) The fibrous product is capable of being regenerated when itsenzymatic activity decreases after the repeated use thereof.

The regeneration of the product of the invention may be carried out asfollows. The product is treated with an aqueous solution containing awater-soluble salt, mineral acid, alkali or their mixtures, and or anaqueous solution containing an oxidizing agent, such as hydrogenperoxide or sodium hypochlorite, whereby the deactivated isomerase isdesorbed or decomposed. Then, the product is again brought into contactwith the glucose isomerase-containing suspension or solution.

The isomerization of glucose into fructose may be carried out in eithera continuous manner or a batchwise manner. The enzymatically activeproduct may be incorporated in a glucose solution while being stirred,or a glucose solution may be passed through a column packed with theenzymatically active product in a fixed bed system.

The separation of fructose from the isomerized glucose solution may becarried out by a conventional procedure. It is, however, preferable toseparate fructose from the isomerized glucose solution by the followingprocedure. That is, the isomerized glucose solution is brought intocontact with zeolite having pores at least 5 angstroms in averagediameter, whereby fructose and glucose contained in the isomerizedglucose solution is adsorbed in the zeolite; and then, the adsorbedfructose is eluted from the zeolite particle.

The zeolite used for the separation of fructose may either by onenaturally occurring or synthesized. The zeolite used is analuminosilicate having a basket structure and represented by theformula: (M_(2/n) O)_(x).(Al₂ O₃)_(y). (SiO₂)_(z).(H₂ O)_(w), where M isa cation, n is a valency of the cation, and x, y, z and w are molenumbers of the respective oxides and water. The zeolite has pores ofrelatively uniform pore diameters, and therefore, is popularly called amolecular sieve. The zeolite used includes, for example, X-type, Y-typeand L-type faujasites (which are supplied by Union Carbide Corp. underthe trade names Zeolite 13X, 10X, SK-40 and SK45) and moldenite (e.g.supplied by Norton Co. under the trade name Zeolon).

The zeolite used should preferably possess pores having an averagediameter of at least 5 angstroms. When the average pore diameter is toosmall, the zeolite exhibits a poor capacity of adsorbing fructose.

The cation of the zeolite may preferably be, for example, univalentalkali metals, such as potassium, sodium, lithium and cesium, andbivalent alkaline earth metals, such as beryllium, magnesium, calcium,strontium and barium. Other metal ions, such as copper, silver, zinc,cadmium, aluminum, lead, iron and cobalt, and ammonium ions, such as NH₄⁺ and NH₃ (CH₃)⁺, may also be used. These cations may be present in thezeolite either alone or in combination.

The ion exchange of the cation of the zeolite may be effected in aconventional manner. For example, sodium zeolite is incorporated in anaqueous 1 N solution of a nitrate of the metal to be exchanged. Themixture is maintained at a temperature of 60° C. for two hours, wherebythe zeolite is infiltrated with the metal nitrate solution. Thisprocedure is repeated several times. Then, the zeolite is completelywashed with deionized water, dried at a temperature of 100° C. for 24hours and, then, heat-treated at a temperature of about 400° C. for twohours.

The zeolite may be used in various forms, including finely dividedparticles and pellets. The pellets may be formed by using a binder forenhancing their mechanical strengths.

The adsorption of fructose and glucose contained in the isomerizedglucose solution may be carried out in a conventional manner. Thezeolite adsorbent may be used, for example, in a fixed bed system, amoving bed system or a fluidized bed system. The adsorption temperaturemay be normal, but an elevated temperature may also be employed forreducing the solution viscosity and enhancing the adsorption rate.However, when the adsorption temperature is too high, fructose andglucose are undesirably degraded. Therefore, the adsorption temperatureshould preferably be lower than about 100° C.

The desorption of the adsorbed fructose and glucose is carried outpreferably by using a desorbent to which the zeolite exhibits anadsorptivity smaller than or equal to that to fructose and glucose, andwhich is capable of dissolving fructose and glucose. A preferabledesorbent includes, for example, water and alcohols, such as methanol,ethanol and their mixtures.

The invention will now be further illustrated by the following examplesin which parts are by weight unless otherwise specified.

In the examples, activity of glucose isomerase, water content,adsorption of albumin and activity efficiency were determined asfollows.

(i) Activity of glucose isomerase

A glucose isomerase-containing specimen was added to an aqueoussubstrate solution containing 0.6 M glucose, 0.01 M MgCl₂.6H₂ O and 0.05M NaHCO₃ and having a pH of 8.2. The mixture was maintained at atemperature of 60° C. for one hour to effect isomerization. Then, theamount of fructose produced was determined by a cystein-carbazolesulfuric acid method or a polarimetry method. The activity of thespecimen was expressed in terms of the unit of U, which corresponded tothe amount of glucose isomerase capable of producing one mg of fructose.

(ii) Activity efficiency (%)

Activity of a glucose isomerase-containing suspension or solution wasdetermined before and after the glucose isomerase immobilizingtreatment. The resulting activity values were referred to as "A" and"A'", respectively. Activity of the immobilizing-treated polymericmaterial was also determined. The resulting activity value was referredto as "B". The activity efficiency was calculated by the equation:##EQU1##

(iii) Water content

A substrate specimen with which glucose isomerase was to be immobilizedwas immersed in a bath of water at room temperature for a sufficientperiod of time to reach saturation. The water clinging to the specimenwithdrawn from the water bath was roughly drained off and then absorbedby an absorbent paper. Immediately thereafter, the specimen was weighed.This water immersion and weighing procedure was repeated twice. Anaverage value for the resulting three weights was referred to as "W".The water content was calculated by the equation: ##EQU2## where W_(o)is the absolute dry weight of the specimen.

(iv) Adsorption of albumin

100 mg of a substrate specimen with which glucose isomerase was to beimmobilized were incorporated in 50 ml of a 0.025 M phosphoric acidbuffer solution, having a pH of 7 and containing 150 mg of albumin. Themixture was stirred at room temperature for three hours. After thespecimen was withdrawn from the mixture, the amount of albumin remainingunadsorbed in the solution was measured.

EXAMPLES 1 THROUGH 9

40 parts of polypropylene (island ingredient) and 60 parts of a blend(sea ingredient) comprised of 49.5 parts of polystyrene, 1.5 parts of alow molecular weight polystyrene, 7.5 parts of polypropylene and 1.5parts of a low molecular weight polypropylene were melt-spun at atemperature of 255° C. into composite filaments having an islands-in-asea type sectional structure (the number of islands=16). The compositefilaments were drawn four times their original length in a conventionalmanner. The filaments had a fineness of 3.7 denier per filament andexhibited a tensile strength of 3.4 g/d and an elongation of 38%.

A blend comprised of 50 parts of polystyrene and 50 parts ofpolypropylene was melt-spun at a temperature of 250° C. into filaments.The blend filaments so obtained were drawn five times their originallength in a conventional manner. The filaments had a fineness of 3.9denier per filament and exhibited a tensile strength of 2.4 g/d and anelongation of 50%.

The respective islands-in-a sea type composite filaments and blendfilaments were β-aminopropionamidomethylated as follows. 1.0 part of thefilaments was incorporated in an acrylamidomethylating solution having acomposition shown in Table I, below. Then, the filament incorporatedsolution was maintained at room temperature for six hours. After beingextracted with methanol, the filaments were treated in a 20% aminesolution in methanol under reflux conditions for two hours. The aminesused are shown in Table I, below. The resulting filaments exhibited atensile strength ranging from 1.0 to 1.5 g/d, and good durability andseparation resistance.

                                      TABLE I                                     __________________________________________________________________________                 Composition of acrylamidomethylaling                                          solution (parts)                                                 Example      Methylol-                                                                           sulfuric                                                                          Nitro-                                                                             Paraform-                                         No.  Filament                                                                              acrylamide                                                                          acid                                                                              benzene                                                                            aldehyde                                                                            Amine                                       __________________________________________________________________________    1    Islands-in-a-sea                                                                      1.0   11.2                                                                              8.8  0     Dimethylamine                                    type composite                                                                filaments                                                                2    Islands-in-a-sea                                                                      1.0   12.4                                                                              7.6  0     Dimethylamine                                    type composite                                                                filaments                                                                3    Islands-in-a-sea                                                                      1.0   12.4                                                                              7.6  0     N,N-dimethylamino-                               type composite               propylamine                                      filaments                                                                4    Islands-in-a-sea                                                                      1.0   12.4                                                                              7.6  0     N,N-diethyl-N'-                                  type composite               methylethylene                                   filaments                    diamine                                     5    Islands-in-a-sea                                                                      1.0   13.8                                                                              6.2  0     Dimethylamine                                    type composite                                                                filaments                                                                6    Islands-in-a-sea                                                                      1.0   10  10   0.010 Dimethylamine                                    type composite                                                                filaments                                                                7    Islands-in-a-sea                                                                      1.0   10  10   0.035 Dimethylamine                                    type composite                                                                filaments                                                                8    Islands-in-a-sea                                                                      1.0   10  10   0.050 Dimethylamine                                    type composite                                                                filaments                                                                9    Blend   1.0   10  10   0     Dimethylamine                                    filaments                                                                __________________________________________________________________________

For comparison purposes, islands-in-a-sea type filaments similar tothose used in EXAMPLE 8 were acrylamidomethylated in a manner similar tothat in EXAMPLE 8. The acrylamidomethylated filaments were treated inconcentrated hydrochloric acid, under reflux conditions, for 20 hours,to be thereby hydrolyzed. Then, the hydrolyzed filaments were treatedwith a formic acid-formalin mixture, to be therebydimethylaminomethylated.

Furthermore, islands-in-a-sea type composite filaments similar to thoseused in Examples 1 through 8 were treated in a solution comprised of 5parts of paraformaldehyde, 25 parts of acetic acid and 70 parts ofconcentrated sulfuric acid, at a temperature of 80° C., for two hours,thereby forming crosslinks therein. The crosslinked filaments weretreated in a solution comprised of 85 parts of chloromethyl ether and 15parts of stannic chloride, at a temperature of 30° C., for one hour, tobe thereby chloromethylated. Then the chloromethylated filaments weretreated in an aqueous 30% trimethylamine solution, to be therebytrimethylammoniummethylated.

Glucose isomerase was immobilized with the respectiveβ-aminopropionamidomethylated, dimethylaminomethylated andtrimethylammoniummethylated filaments as follows. Glucose isomerase wasextracted from "Glucose Isomerase Nagase" (Streptomycesphaeochromogenus, supplied by Nagase Sangyo K.K.). The extract solutionwas centrifugated to obtain a glucose isomerase extract exhibiting anactivity of 3,000 U/20 ml and having a pH of 8. 100 mg of the filamentswere incorporated in 20 ml of the glucose isomerase extract, and theobtained mixture was stirred at room temperature for six hours, wherebyglucose isomerase was immobilized with the filaments. The filaments usedfor the isomerase immobilization and the isomerase immobilized filamentshad the characteristics shown in Table II, below.

                                      TABLE II                                    __________________________________________________________________________                                            Activity of                                           Amount of                                                                           Form of           isomerase                                             functional                                                                          functional                                                                              Albumin immobilized                           Example                                                                            Exchange   group group Water                                                                             adsorption*.sup.3                                                                     filaments                             No.  group      (meq/g)                                                                             *1    content                                                                           (mg/g filament)                                                                       (U/100 mg)                            __________________________________________________________________________    1    β-aminopropion-                                                                     2.7   Free  2.8 --      2600                                       amidemethyl                                                                   group                                                                    2    β-aminopropion-                                                                     2.6   "     1.3 200     1360                                       amidemethyl                                                                   group                                                                    3    β-aminopropion-                                                                     2.0   "     1.8 --      680                                        amidemethyl                                                                   group                                                                    4    β-aminopropion-                                                                     2.0   "     2.1 --      1330                                       amidemethyl                                                                   group                                                                    5    β-aminopropion-                                                                     1.8   "     1.0 --      850                                        amidemethyl                                                                   group                                                                    6    β-aminopropion-                                                                     2.9   "     2.1 570     2370                                       amidemethyl                                                                   group                                                                    7    β-aminopropion-                                                                     3.0   "     1.7 --      1410                                       amidemethyl                                                                   group                                                                    8    β-aminopropion-                                                                     3.1   "     1.5 450     830                                        amidemethyl                                                                   group                                                                    9    β-aminopropion-                                                                     2.9   "     1.3 130     640                                        amidemethyl                                                                   group                                                                    *2                                                                            Com. 1                                                                             Dimethylamino-                                                                           3.5   Free  1.5 600     100                                   Com. 2                                                                             methyl group                                                                             3.1   Cl    5.1 600     250                                   Com. 3                                                                             Trimethylammonium-                                                                       2.7   Cl    2.5 --      500                                        methyl group                                                             __________________________________________________________________________     *1 "Free" refers to a free form functional group which was formed by          treating the filaments in an aqueous 1N sodium hydroxide solution and,        then, washing the treated filaments with deionized water. "Cl" refers to      chloride form functional group which was formed by treating the filaments     in an aqueous 1N hydrochloric acid solution and, then, wasing the treated     filaments with deionized water.                                               *2 "Com" refers to Comparative Example.                                       *3 The filaments used for the isomerase immobilization exhibited a            tendency of adsorbing invertase and catalase, which was similar to their      tendency of adsorbing albumin.                                           

The activity efficiency of the isomerase immobilized filaments of theinvention was high, i.e., in the range of from about 80 to 90%.

The following will be seen from Table II. First, the filaments used inthe invention have a capacity of immobilizing a large amount of glucoseisomerase and, thus, the isomerase immobilized filaments exhibit anenhanced activity per unit weight of the filaments. Secondly, althoughthe filaments used in the invention have a low water content, theyexhibit an enhanced activity. This is particularly true where thefilaments have little crosslinked structure (Examples No. 1 through 5).Furthermore, in the case where the filaments possess the same functionalgroups and the same crosslinked structure, the larger the amount of thefunctional groups, the higher the activity of the isomerase immobilizedfilaments. In the case where the filaments possess the same functionalgroups and the same amounts of the functional groups, the lower thedegree of crosslinking, the higher the activity of the isomeraseimmobilized filaments. Also, an organic amine containing a secondaryamino group results in the isomerase immobilized filaments exhibiting ahigher activity than that of the filaments prepared by using an organicamine containing a primary amino group.

EXAMPLE 10

Islands-in-a-sea type composite filaments similar to those used inExamples 1 through 8 were knitted into a tubular fabric. The tubularknitted fabric was β-aminopropionamidomethylated in a manner similar tothat mentioned in Example 2. The resultant fabric had the followingcharacteristics. The amount of the functional group was 2.8 meq/g. Thewater content (in the free form) was 1.9. The albumin adsorption was 240mg/g.

A glucose isomerase extract exhibiting an activity of 3,000 U/20 ml wasprepared in a manner similar to that mentioned in Examples 1 through 9.The pH of the isomerase extract was adjusted to a value shown in TableIII, below, by adding thereto an aqueous 1 N sodium hydroxide solutionor an aqueous 1 N hydrochloric acid solution. 100 mg of theabove-mentioned knitted fabric having the functional groups in a freeform were incorporated in 20 ml of the isomerase extract and theobtained mixture was stirred at room temperature, for six hours, wherebyglucose isomerase was immobilized. The resultant fabric had thecharacteristics shown in Table III, below.

                  TABLE III                                                       ______________________________________                                                      Activity of                                                                   Isomerase                                                       pH of         immobilized                                                                              Activity                                             isomerase     fabric     efficiency                                           extract       (U/100 mg) (%)                                                  ______________________________________                                        7             710        80                                                   8             850        81                                                   9             1010       80                                                   10            1290       77                                                   11            500        81                                                   ______________________________________                                    

As will be seen from Table III, a glucose isomerase extract having a pHof below 10 results in an isomerase immobilized fabric exhibiting a highactivity.

EXAMPLE 11

100 mg of a knitted fabric having the functional groups in a free form,which fabric was similar to that mentioned in Example 10, wereincorporated in 10 ml of a glucose isomerase extract having an activityof 1,500 U and a pH of 8, which extract was similar to that used inExamples 1 through 9. The mixture was stirred at room temperature, forsix hours, whereby glucose isomerase was immobilized. The isomeraseimmobilized fabric exhibited an activity of 935 U/100 mg. The activityefficiency was 81%. Thereafter, the isomerase immobilized fabric wastreated in 10 ml of an aqueous 0.1% glutaraldehyde solution having a pHof 8, at room temperature, for 30 minutes. The resultant fabricexhibited an activity of 900 U/100 mg. The activity efficiency was 78%.

The glutaraldehyde treated fabric was repeatedly used for glucoseisomerization. When the fabric was used ten times, it retained 90% ofits initial activity. Similarly, the isomerase immobilizedglutaraldehyde-untreated fabric was repeatedly used for glucoseisomerization. When the fabric was used ten times, it retained 85% ofits initial activity.

For comparison purposes, glucose isomerase was immobilized with acommercially available ion exchange resin, Amberlite IRA-904, havingtrimethylammoniummethyl groups in a chloride form, in a manner similarto that mentioned above. The isomerase immobilized resin exhibited anactivity of 450 U/100 mg. The activity efficiency was 75%. This resinretained 65% of its initial activity after ten repeated isomerizations.Similarly, the isomerase immobilized filaments obtained in ComparativeExample 3 were repeatedly used for glucose isomerization. The filamentsalso retained 65% of their initial activity after ten repeatedisomerizations.

The above results show that the enzymatically active product of theinvention is stable in a substrate solution and, hence, exhibits a goodretention of activity. Particularly, the product treated with amulti-functional proteinaceous crosslinking agent, i.e., glutaraldehyde,exhibits an excellent retention of activity.

EXAMPLE 12

100 mg of a knitted fabric having the functional groups in a free form,similar to that mentioned in Example 10, were incorporated in 10 ml of aglucose isomerase extract having an activity of 1,500 U and a pH of 8,similar to that used in Examples 1 through 9. The mixture was stirred atroom temperature, for six hours, whereby glucose isomerase wasimmobilized. The isomerase immobilized fabric exhibited an activity of935 U/100 mg. The activity efficiency was 81%.

The isomerase immobilized fabric was treated in a substrate solution ata temperature of 90° C., for 10 minutes, whereby the isomerase wasdeactivated. Then, the fabric was treated in an aqueous 1 M sodiumchloride solution, while being stirred, at room temperature, for onehour. Then, the fabric was treated in an aqueous 1 N sodium hydroxidesolution, whereby the functional groups were converted to a free form.Thereafter, the fabric was again treated with a glucose isomeraseextract in a manner similar to that mentioned above, whereby theisomerase was immobilized. The isomerase immobilized fabric exhibited anactivity of 920 U/100 mg. The activity efficiency was 80%.

The above results show that, when the enzymatic activity of the productof the invention is reduced, the product can be regenerated by removingthe deactivated enzyme therefrom and then immobilizing the isomerasewith the product.

EXAMPLE 13

1 g of a knitted fabric having the functional groups in a free form,similar to that mentioned in Example 10, was incorporated in 75 ml of aglucose isomerase extract suspension containing ruptured cell pieces andexhibiting an activity of 15,000 U. The mixture was stirred at roomtemperature, for six hours, whereby glucose isomerase was immobilized.The separation of the isomerase immobilized fabric from the rupturedcell-containing suspension could be easily carried out. Thereafter, theisomerase immobilized fabric was treated in 100 ml of an aqueous 0.1%glutaraldehyde solution having a pH of 8, at room temperature, for 30minutes. The resultant fabric exhibited an activity of 12,500 U. Theactivity efficiency was 84%. Even when the fabric was treated in anaqueous 1 M sodium chloride solution having a pH of 8, its enzymaticactivity was reduced only to a negligible extent.

EXAMPLE 14

A felt was manufactured from islands-in-a sea type composite filaments,which were prepared in a manner similar to that mentioned in Examples 1through 8, but at a drawing ratio of 3.5 in place of 4.0. The felt wasβ-aminopropionamidomethylated in a manner similar to that employed inExample 2. The resultant fabric contained 2.8 meq of theβ-aminopropionamidomethyl group per g of the fabric and exhibited awater content of 1.9 in a free form of the functional group. Then, thefelt was cut into a circular shape. 1.3 g of the circular felt werepacked in a column, 1.6 cm in diameter, equipped with a heater. 200 mlof a glucose isomerase extract having an activity of 30,000 U and a pHof 8 were repeatedly passed through the felt-packed column at atemperature of 50° C., for six hours, whereby glucose isomerase wasimmobilized. The glucose isomerase extract used exhibited an activity of960 U after the immobilization treatment. Then, an aqueous substratesolution containing 3 M glucose and 0.005 M magnesium sulfate and havinga pH of 8 was passed through the column to isomerize glucose. Thepercentage isomerization was 40% and 45% at flow rates of 75 ml/hr and55 ml/hr, respectively.

For comparison purposes, 4.5 g of a commercially available ion exchangeresin, Amberlite IRA-904, of an SO₄ - form were incorporated in 200 mlof a glucose isomerase extract having an activity of 30,000 U and a pHof 8. The mixture was stirred at a temperature of 50° C., for six hours,whereby glucose isomerase was immobilized. The glucose isomerase extractused exhibited an activity of 7,800 U after the immobilizationtreatment. The isomerase immobilized resin were packed in a column 1.6cm in diameter equipped with a heater. An aqueous substrate solutionsimilar to that mentioned above was passed through the ion exchangeresin packed column at a temperature of 60° C. The percentageisomerization was 40% and 45% at flow rates of 48 ml/hr and 35 ml/hr,respectively.

Furthermore, 10 g of Sweetzyme (trade name for immobilized glucoseisomerase supplied by Novo Industri A/S, Denmark) were packed in acolumn, 1.6 cm in diameter, equipped with a heater. A substrate solutionsimilar to that mentioned above was passed through the column at atemperature of 60° C. The percentage isomerization was 40% and 45% atflow rates of 53 ml/hr and 37 ml/hr, respectively.

The above mentioned results show that the enzymatically active productof the invention exhibits a high activity per unit weight of the productand, consequently, the isomerization of glucose can be effected with ahigh productivity by passing a substrate solution at an enhanced ratethrough the active product-packed column.

EXAMPLE 15

100 g of a Y-type zeolite (Y-K, its metal ion was a potassium ion)having a particle size of from 20 to 40 meshes were packed in a columnhaving an inner diameter of 15 mm. The packed zeolite had a height of 92cm. The isomerized glucose solution obtained in Example 14 andcontaining 0.55 g of glucose and 0.45 of fructose was continuouslysupplied into the top of the zeolite packed column at room temperatureand at a flow rate of 33 ml/hr, whereby the solution was developed inthe column. The effluent withdrawn from the column bottom was sampled atintervals, and it was found that glucose was initially withdrawn and,then, fructose was withdrawn. Analysis of 35 ml of the effluentfractions ranging from 125 ml to 160 ml in total volume of effluentshowed that the effluent fractions contained no fructose.

EXAMPLE 16

An isomerized glucose solution obtained by the procedure mentioned inExample 14, and containing 0.6 g of glucose and 0.4 g of fructose, wassubjected to adsorption chromatography in a manner similar to thatmentioned in Example 15. Analysis of the effluent showed that glucosewas initially withdrawn and, then, fructose was withdrawn. Thefructose-containing fractions were separated, concentrated and, then,crystallized in a conventional manner, whereby a fructose crystal couldbe obtained.

What we claim is:
 1. A process for isomerizing to produce fructose fromglucose, comprising bringing a glucose-containing solution into contactwith an enzymatically active product comprising an organic polymericmaterial comprised of at least 50% by weight, based on the weight of theorganic polymeric material, of a monovinyl aromatic compound inpolymerized form, said polymerized monovinyl aromatic compound having aβ-aminopropionamidomethyl group as a side chain represented by theformula I: ##STR11## wherein R₁ is selected from hydrogen, an alkylgroup having 1 to 6 carbon atoms and a hydroxyalkyl group having 2 to 6carbon atoms, R₂ is selected from an alkyl group having 1 to 6 carbonatoms, a hydroxyalkyl group having 2 to 6 carbon atoms, ##STR12## (whereX, Z₁ and Z₂ are selected from hydrogen and an alkyl group having 1 to 6carbon atoms, and n is an integer of from 2 to 6), or R₁ and R₂ formtogether with the nitrogen atom, to which R₁ and R₂ are bonded, aheterocyclic structure represented by the formula: ##STR13## where A is--CH₂ --, --O-- or --NR₆ -(wherein R₆ is H or an alkyl group having 1 to6 carbons), and R₃, R₄ and R₅ may be the same as or different from eachother and are selected from hydrogen and a methyl group, said organicpolymeric material having glucose isomerase immobilized with said sidechain of the organic polymeric material, whereby at least a part of theglucose is isomerized into fructose.
 2. The process according to claim1, wherein the content of the β-aminopropionamidomethyl group of theformula I is from about 0.5 to 5.0 meq/g of the organic polymericmaterial.
 3. The process according to claim 1, wherein the content ofthe β-aminopropionamidomethyl group of the formula I is from about 2.0to 5.0 meq/g of the organic polymeric material.
 4. The process accordingto claim 1, wherein the amount of the immobilized glucose isomerasecorresponds to the activity of from 2,000 to 50,000 U per g of theenzymatically active product.
 5. The process according to claim 1,wherein the monovinyl aromatic compound is at least one compoundselected from the group consisting of styrene, α-methylstyrene,vinyltoluene, vinylxylene and chlorostyrene.
 6. The process according toclaim 1 wherein the enzymatically active product is in the form of afiber, a particulate or a film.
 7. The process according to claim 6,wherein said product is a fiber, and wherein said fiber comprises, inaddition to said organic polymeric material predominantly comprised ofthe polymerized monovinyl aromatic compound having theβ-aminopropionamidomethyl group, a fiber-forming organic polymericmaterial.
 8. The process according to claim 6, wherein said product is afiber, and wherein said fiber is a core-sheath type or islands-in-a seatype composite fiber, the core or island ingredient being comprised ofsaid fiber-forming organic polymeric material and the sheath or seaingredient being predominantly comprised of said organic polymericmaterial predominantly comprised of a polymerized monovinyl aromaticcompound having the β-aminopropionamidomethyl group.
 9. The processaccording to claim 6, wherein said product is a fiber, and wherein saidfiber is comprised of a mixture of said fiber-forming organic polymericmaterial and said organic polymeric material predominantly comprised ofthe polymerized monovinyl aromatic compound having theβ-aminopropionamidomethyl group.
 10. The process according to claim 7, 8or 9, wherein said fiber is in the form of non-woven fabric, paper,woven fabric or knitted fabric.
 11. The process according to claim 7, 8or 9, wherein, said fiber forming organic polymeric material is at leastone polymer selected from the group consisting of polyolefins,polyamides, polyesters and their copolymers.
 12. The process accordingto claim 1, 7, 8 or 9, wherein the glucose isomerase is cross-linkedwith a crosslinking agent capable of crosslinking a protein.
 13. Theprocess according to claim 12, wherein the crosslinking agent is atleast one compound selected from the group consisting of glutaraldehyde,dialdehyde starch, tolylene diisocyanate and hexamethylene diisocyanate.